Antisense inhibition of PTEN expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of PTEN. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding PTEN. Methods of using these compounds for modulation of PTEN expression and for treatment of diseases and conditions associated with expression of PTEN are provided. Such conditions include diabetes and hyperproliferative conditions. Methods for decreasing blood glucose levels, inhibiting PEPCK expression, decreasing blood insulin levels, decreasing insulin resistance, increasing insulin sensitivity, decreasing blood triglyceride levels or decreasing blood cholesterol levels in an animal using the compounds of the invention are also provided. The animal is preferably a human; also preferably the animal is a diabetic animal.

This application is a continuation-in-part of PCT applicationPCT/US99/29594, filed Dec. 14, 1999, which is a continuation of U.S.patent application Ser. No. 09/358,381, filed Jul. 21, 1999, now issuedas U.S. Pat. No. 6,020,199.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of PTEN. In particular, this invention relates toantisense compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding human PTEN. Sucholigonucleotides have been shown to modulate the expression of PTEN.

BACKGROUND OF THE INVENTION

One of the principal mechanisms by which cellular regulation is effectedis through the transduction of extracellular signals across the membranethat in turn modulate biochemical pathways within the cell. Proteinphosphorylation represents one course by which intracellular signals arepropagated from molecule to molecule resulting finally in a cellularresponse. These signal transduction cascades are tightly regulated andoften overlap as evidenced by the existence of multiple protein kinaseand phosphatase families and isoforms.

Because phosphorylation is such a ubiquitous process within cells andbecause cellular phenotypes are largely influenced by the activity ofthese pathways, it is currently believed that a number of disease statesand/or disorders are a result of either aberrant activation orfunctional mutations in the molecular components of these cascades.Consequently, considerable attention has been devoted to thecharacterization of proteins exhibiting either kinase or phosphataseenzymatic activity.

PTEN (also known as MMAC1 and TEP1) is a dual-specificity proteinphosphatase recently implicated as a phosphoinositide phosphatase in theinsulin-signaling pathway. In studies of human 293 cells, PTEN was shownto dephosphorylate phosphatidylinositol 3,4,5-triphosphate (PIP3), anacidic lipid that is involved in cellular growth signaling (Maehama andDixon, J. Biol. Chem., 1998, 273, 13375-13378). In Drosophila, studiesof PTEN activation and overexpression demonstrated that PTEN affectsboth cell size and cell cycle progression during eye development. Inaddition, the authors demonstrated that PTEN acts in the insulinsignaling pathway and that all signals from the insulin receptor can beantagonized by PTEN. These data suggest that modulation of PTEN mayrepresent a means for modulating altered insulin signaling (Huang etal., Development, 1999, 126, 5365-5372).

PIP3 is an important second messenger generated specifically by theactions of phosphatidylinositol 3-kinase (PI3-kinase) following insulinbinding (Stephens et al., Science, 1998, 279, 710-714). overexpressionof PTEN was shown to reduce the levels of PIP3 in insulin treated cellswithout affecting the activity of PI3-kinase (Maehama and Dixon, J.Biol. Chem., 1998, 273, 13375-13378). These results establish a role forPTEN as a regulator of the downstream pathways initiated by insulinbinding. In the nematode, Caenorhabditis elegans, the PTEN homolog,daf-18, has been cloned and shown to antagonize signaling cascadesassociated with PI3-kinase (Gil et al., Proc. Natl. Acad. Sci. USA,1999, 96, 2925-2930). The authors suggest that this may indicate thatPTEN may play a role in mammalian glucose homeostasis, and that PTEN maybe a rational pharmacological target for Type II diabetes.

The PTEN protein also contains an amino terminal domain homologous totensin and auxilin, proteins that interact with actin filaments and areinvolved in synaptic vesicle transport, respectively (Li and Sun, CancerRes., 1997, 57, 2124-2129; Li et al., Science, 1997, 275, 1943-1947;Steck et al., Nat. Genet., 1997, 15, 356-362). In addition, PTEN is alsodownregulated by transforming growth factor beta (TGF-β), a cytokineinvolved in the regulation of cell adhesion and motility (Li and Sun,Cancer Res., 1997, 57, 2124-2129). Taken together these data suggestthat PTEN plays a dual role within the cell by mediating the activity ofprotein kinases while regulating cell motility (Tamura et al., Science,1998, 280, 1614-1617).

Finally, a large number of naturally occurring point and germ-linemutations have been identified in PTEN. These mutations have beenisolated from several cancerous solid tumors and cell lines includingbrain, breast, prostate, ovary, skin, thyroid, lung, bladder and colon(Teng et al., Cancer Res., 1997, 57, 5221-5225) and have led to theclassification of PTEN as a tumor suppressor gene. Disclosed in the PCTpublication WO 99/02704 are PTEN proteins and altered PTEN proteins andthe nucleic acids encoding them. Also disclosed are methods of diagnosisand treatment utilizing compositions comprising PTEN or altered PTENproteins or nucleic acid molecules.

The most common mutations found in tumor specimens were frameshiftmutations (1 in 17 breast carcinomas), missense variants (1 in 10melanomas), nonsense mutations and splice variants (2 in 5 pediatricglioblastomas). In tumor cell lines exhibiting loss of heterozygosity(LOH), 11 homozygous deletions affecting the coding region weredetected. Two cell lines had lost all 9 exons and nine cell lines hadhomozygous deletions of portions of the coding regions. The remaining 65cell lines contained 3 frameshift, one nonsense and 8 nonconservativemissense mutations (Teng et al., Cancer Res., 1997, 57, 5221-5225).

The known germ-line mutations in PTEN give rise to three distinctautosomal dominant disorders known as Cowden disease (CD) (Liaw et al.,Nat. Genet., 1997, 16, 64-67; Nelen et al., Hum. Mol. Genet., 1997, 6,1383-1387; Tsou et al., Hum. Genet., 1998, 102, 467-473),Lhermitte-Duclos disease (LDD) (Liaw et al., Nat. Genet., 1997, 16,64-67) and Bannayan-Zonana syndrome (BZS, also known asBannayan-Riley-Ruvalcaba syndrome, Ruvalcaba-Myhre-Smith syndrome andRiley-Smith syndrome) (Arch et al., Am. J. Med. Genet., 1997, 71,489-493; Marsh et al., Nat. Genet., 1997, 16, 333-334). All of theseconditions are characterized by the presence of gastrointestinal polyps,increased tumor susceptibility and developmental defects.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of PTEN, and strategies aimed at inhibiting and/orinvestigating PTEN function have involved the use of gene knock-outs inmice and ribozyme- and vector-based antisense-mediated regulation ofPTEN expression.

Di Cristofano et al. demonstrated that the complete disruption of themouse PTEN gene by homologous recombination resulted in embryoniclethality (Di Cristofano et al., Nat. Genet., 1998, 19, 348-355). Bycontrast, PTEN +/− chimeric mice were phenotypically identical to theirwild-type littermates. However, post-mortem analysis revealed abnormalpathological conditions similar to those observed in human diseases.

Other studies involving the targeted disruption of exons 3 and 5 in micedemonstrated that homozygous mice died by day 9.5 of development andthat immortalized cells from these embryos showed decreased sensitivityto various apoptotic stimuli (Stambolic et al., Cell, 1998, 95, 29-39).These cells also displayed constitutively elevated activity of thePKB/Akt kinases. Taken together these results suggest that PTEN acts bynegatively regulating the PI3-kinase/PKB/Akt pathway.

Devlin and Clawson identified ribozyme-accessible sites on full lengthPTEN cDNA and, using these results, designed a ribozyme construct forthe purpose of regulating PTEN transcripts. Proc. Am. Assoc. CancerRes., 1999, 40, 438.

Tamura et al. established stable transfectant lines of mouse 3T3 cellsin which the expression of PTEN was up- or down-regulated usingexpression plasmids containing full length sense PTEN or full-lengthantisense PTEN. The antisense construct enhanced cell migration.Science, 1998, 280, 1614-1617.

There remains a long felt need for agents capable of effectivelyinhibiting PTEN function and antisense technology is emerging as aneffective means for reducing the expression of specific gene products.This technology may therefore prove to be uniquely useful in a number oftherapeutic, diagnostic, and research applications for the modulation ofPTEN expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly antisenseoligonucleotides, which are targeted to a nucleic acid encoding PTEN,and which modulate the expression of PTEN. Pharmaceutical and othercompositions comprising the antisense compounds of the invention arealso provided. Further provided are methods of modulating the expressionof PTEN in cells or tissues comprising contacting said cells or tissueswith one or more of the compounds or compositions of the invention.Further provided are methods of treating an animal, particularly ahuman, suspected of having or being prone to a disease or conditionassociated with expression of PTEN by administering a therapeutically orprophylactically effective amount of one or more of the antisensecompounds or compositions of the invention. Such conditions includediabetes and hyperproliferative conditions. Methods for decreasing bloodglucose levels, inhibiting PEPCK expression, decreasing blood insulinlevels, decreasing insulin resistance, increasing insulin sensitivity,decreasing blood triglyceride levels or decreasing blood cholesterollevels in an animal using the compounds of the invention are alsoprovided. The animal is preferably a human; also preferably the animalis a diabetic animal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding PTEN, ultimately modulating the amountof PTEN produced. This is accomplished by providing antisense or otheroligonucleotide compounds which specifically hybridize with one or morenucleic acids encoding PTEN. As used herein, the terms “target nucleicacid” and “nucleic acid encoding PTEN” encompass DNA encoding PTEN, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of PTEN. Inthe context of the present invention, “modulation” means either anincrease (stimulation) or a decrease (inhibition) in the expression of agene. In the context of the present invention, inhibition is thepreferred form of modulation of gene expression and mRNA is a preferredtarget.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or MRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding PTEN. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding PTEN, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 31′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an MRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense and other compounds of the invention which hybridize to thetarget and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are herein referredto as “active sites” and are therefore preferred sites for targeting.While not wishing to be bound by theory, it is believed that the activesites so identified are particularly suitable for ligand binding, due toaccessibility or other reasons. Therefore another embodiment of theinvention encompasses compounds which hybridize to these active sites.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Oligonucleotide drugs, both antisense andribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric compounds,including but not limited to oligonucleotide mimetics such as aredescribed below. The compounds in accordance with this inventionpreferably comprise from about 8 to about 50 nucleobases (i.e. fromabout 8 to about 50 linked nucleosides), and even more preferably fromabout 12 to about 30 nucleobases. The present invention is also intendedto comprehend other oligomeric compounds from about 8 to about 50nucleobases in length which hybridize to the nucleic acid target andwhich inhibit expression of the target. Such compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred compounds useful in this inventioninclude antisense oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH)₃—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference, and U.S. Pat. No. 5,750,692, which iscommonly owned with the instant application and also herein incorporatedby reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The compounds in accordance with this invention may be conveniently androutinely made through the well-known technique of solid phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and alkylatedderivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to he methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of PTEN is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingPTEN, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding PTEN can be detected by meansknown in the art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of PTEN in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1andWO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, N.Y., 1996, pp.934-935). Various natural bile salts, and their synthetic derivatives,act as penetration enhancers. Thus the term “bile salts” includes any ofthe naturally occurring components of bile as well as any of theirsynthetic derivatives. The bile salts of the invention include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling, Va.) Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling, Va. or ChemGenes,Needham, Mass.).

2′-Fluoro amidites

2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT3′phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylurine (96 g,10 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POC₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethylazodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]5-methyluridine

2′O-([2-phthalimidoxy)ethyl]5′-t-butyldiphenylsilyl-5-methyluridine (3.1g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine(300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h themixture was filtered, the filtrate was washed with ice cold CH₂Cl₂ andthe combined organic phase was washed with water, brine and dried overanhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 240 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

20′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]5-methyluridine

2[2-(Dimethylamino)ethoxy] ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]5-methyluridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P=O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or U.S. Pat. No. 5,625,050, hereinincorporated by reference.

Phosphoramidite oligonucleotides are prepared as described in U.S.Patent, U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, hereinincorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]—[2′ -deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl)Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing four cell types are provided for illustrative purposes, butother cell types can be routinely used.

T-24 Cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinely.maintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10

Analysis of Oligonucleotide Inhibition of PTEN Expression

Antisense modulation of PTEN expression can be assayed in a variety ofways known in the art. For example, PTEN mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+ MRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions. Other methods of PCR are also known in the art.

PTEN protein levels can be quantitated in a variety of ways well knownin the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to PTEN can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc.,. Valencia Calif.). Essentiallyafter lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

Real-time Quantitative PCR Analysis of PTEN mRNA Levels

Quantitation of PTEN mRNA levels was determined by realtime quantitativePCR using the ABI PRISM™ 7700 Sequence Detection System (PE-AppliedBiosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, obtained from either Operon Technologies Inc.,Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). PTEN probes and primers were designedto hybridize to the human PTEN sequence, using published sequenceinformation (GenBank accession number U93051, incorporated herein as SEQID NO:1).

For PTEN the PCR primers were:

forward primer: AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2) reverseprimer: TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3) and the PCR probe was:FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 5) reverse primer:GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 7) where JOE (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

Northern Blot Analysis of PTEN mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions with a PTEN specific probe prepared by PCR usingthe forward primer AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2) and thereverse primer TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.). Hybridized membranes were visualizedand quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDHlevels in untreated controls.

Example 15

Antisense Inhibition of PTEN Expression-phosphorothioateOligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human PTEN RNA, usingpublished sequences (GenBank accession number U93051, incorporatedherein as SEQ ID NO: 1). The oligonucleotides are shown in Table 1.Target sites are indicated by the first (5′ most) nucleotide number, asgiven in the sequence source reference (Genbank accession no. U93051),to which the oligonucleotide binds. All compounds in Table 1 areoligodeoxynucleotides with phosphorothioate backbones (internucleosidelinkages) throughout. The compounds were analyzed for effect on PTENmRNA levels by quantitative real-time PCR as described in other examplesherein. Data are averages from two experiments. If present, “N.D.”indicates “no data”.

TABLE 1 Inhibition of PTEN mRNA levels by phosphorothioateoligodeoxynucleotides TARGET % SEQ ID ISIS# REGION SITE SEQUENCEInhibition NO. 29534 Coding 19 cgagaggcggacgggacc 0 8 29535 Coding 57cgggcgcctcggaagacc 62 9 29536 Coding 197 tggctgcagcttccgaga 73 10 29537Coding 314 cccgcggctgctcacagg 81 11 29538 Coding 421 caggagaagccgaggaag51 12 29539 Coding 494 gggaggtgccgccgccgc 42 13 29540 Coding 581atggtgacaggcgactca 75 14 29541 Coding 671 ccgggtccctggatgtgc 76 15 29542Coding 757 cctccgaacggctgcctc 60 16 29543 Coding 817 tctcctcagcagccagag34 17 29544 Coding 891 cgcttggctctggaccgc 84 18 29545 Coding 952tcttctgcaggatggaaa 0 19 29546 Coding 1048 tgctaacgatctctttga 43 20 29547Coding 1106 ggataaatataggtcaag 0 21 29548 Coding 1169 tcaatattgttcctgtat0 22 29549 3′ UTR 1262 ttaaatttggcggtgtca 0 23 29550 3′ UTR 1342caagatcttcacaaaagg 0 24 29551 3′ UTR 1418 attacaccagttcgtccc 59 25 295523′ UTR 1504 tgtctctggtccttactt 34 26 29553 3′ UTR 1541acatagcgcctctgactg 72 27 29554 3′ UTR 1606 tgtgaaacaacagtgcca 75 2829555 3′ UTR 1694 gaatatatcttcaccttt 42 29 29556 3′ UTR 1792ggaagaactctactttga 38 30 29557 3′ UTR 1855 tgaagaatgtatttaccc 44 3129558 3′ UTR 1916 atttcttgatcacataga 0 32 29559 3′ UTR 2020ggttggctttgtctttat 77 33 29560 3′ UTR 2098 tgctagcctctggatttg 74 3429561 3′ UTR 2180 tctggatcagagtcagtg 44 35 29562 3′ UTR 2268tattttcatggtgtttta 76 36 29563 3′ UTR 2347 tgttcctataactggtaa 58 3729564 3′ UTR 2403 gtgtcaaaaccctgtgga 72 38 29565 3′ UTR 2523actggaataaaacgggaa 15 39 29566 3′ UTR 2598 acttcagttggtgacaga 69 4029567 3′ UTR 2703 tagcaaaacctttcggaa 51 41 29568 3′ UTR 2765aattatttcctttctgag 14 42 29569 3′ UTR 2806 taaatagctggagatggt 55 4329570 3′ UTR 2844 cagattaataactgtagc 9 44 29571 3′ UTR 2950ccccaatacagattcact 52 45 29572 3′ UTR 3037 attgttgctgtgtttctt 64 4629573 3′ UTR 3088 tgtttcaagcccattctt 65 47

As shown in Table 1, SEQ ID NOs 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,20, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38, 40, 41, 43, 45,46 and 47 demonstrated at least 30% inhibition of PTEN expression inthis assay and are therefore preferred. The target sites to which thesepreferred sequences are complementary are herein referred to as “activesites” and are therefore preferred sites for targeting by compounds ofthe present invention.

Example 16

Antisense Inhibition of PTEN Expression-phosphorothioate 2′-MOE GapmerOligonucleotides

In accordance with the present invention, a second series ofoligonucleotides targeted to human PTEN were synthesized. Theoligonucleotide sequences are shown in Table 2. Target sites areindicated by the first (5′ most) nucleotide number, as given in thesequence source reference (Genbank accession no. U93051), to which theoligonucleotide binds.

All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 18nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by fournucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide.Cytidine residues in the 2′-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from two experiments. If present,“N.D.” indicates “no data”.

TABLE 2 Inhibition of PTEN mRNA levels by chimeric pho- sphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET % SEQ IDISIS# REGION SITE SEQUENCE Inhibition NO. 29574 Coding 19cgagaggcggacgggacc 71 8 29575 Coding 57 cgggcgcctcggaagacc 37 9 29576Coding 197 tggctgcagcttccgaga 76 10 29577 Coding 314 cccgcggctgctcacagg86 11 29578 Coding 421 caggagaagccgaggaag 71 12 29579 Coding 494gggaggtgccgccgccgc 85 13 29580 Coding 581 atggtgacaggcgactca 0 14 29581Coding 671 ccgggtccctggatgtgc 20 15 29582 Coding 757 cctccgaacggctgcctc82 16 29583 Coding 817 tctcctcagcagccagag 85 17 29584 Coding 891cgcttggctctggaccgc 92 18 29585 Coding 952 tcttctgcaggatggaaa 72 19 29586Coding 1048 tgctaacgatctctttga 79 20 29587 Coding 1106ggataaatataggtcaag 61 21 29588 Coding 1169 tcaatattgttcctgtat 52 2229589 3′ UTR 1262 ttaaatttggcggtgtca 82 23 29590 3′ UTR 1342caagatcttcacaaaagg 0 24 29591 3′ UTR 1418 attacaccagttcgtccc 77 25 295923′ UTR 1504 tgtctctggtccttactt 79 26 29593 3′ UTR 1541acatagcgcctctgactg 83 27 29594 3′ UTR 1606 tgtgaaacaacagtgcca 73 2829595 3′ UTR 1694 gaatatatcttcaccttt 0 29 29596 3′ UTR 1792ggaagaactctactttga 0 30 29597 3′ UTR 1855 tgaagaatgtatttaccc 84 31 295983′ UTR 1916 atttcttgatcacataga 5 32 29599 3′ UTR 2020 ggttggctttgtctttat60 33 29600 3′ UTR 2098 tgctagcctctggatttg 86 34 29601 3′ UTR 2180tctggatcagagtcagtg 82 35 29602 3′ UTR 2268 tattttcatggtgtttta 58 3629603 3′ UTR 2347 tgttcctataactggtaa 49 37 29604 3′ UTR 2403gtgtcaaaaccctgtgga 62 38 29605 3′ UTR 2523 actggaataaaacgggaa 22 3929606 3′ UTR 2598 acttcagttggtgacaga 79 40 29607 3′ UTR 2703tagcaaaacctttcggaa 52 41 29608 3′ UTR 2765 aattatttcctttctgag 67 4229609 3′ UTR 2806 taaatagctggagatggt 37 43 29610 3′ UTR 2844cagattaataactgtagc 35 44 29611 3′ UTR 2950 ccccaatacagattcact 0 45 296123′ UTR 3037 attgttgctgtgtttctt 0 46 29613 3′ UTR 3088 tgtttcaagcccattctt43 47

As shown in Table 2, SEQ ID NOs 8, 9, 10, 11, 12, 13, 16, 17, 18, 19,20, 21, 22, 23, 25, 26, 27, 28, 31, 33, 34, 35, 36, 37, 38, 40, 41, 42,43, 44 and 47 demonstrated at least 30% inhibition of PTEN expression inthis experiment and are therefore preferred. The target sites to whichthese preferred sequences are complementary are herein referred to as“active sites” and are therefore preferred sites for targeting bycompounds of the present invention.

Example 17

Western Blot Analysis of PTEN Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to PTEN is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 18

Antisense Inhibition of PTEN Expression-dose Response in Human, Mouseand Rat Hepatocytes

In accordance with the present invention, two additionaloligonucleotides targeted to human PTEN were designed and synthesized.ISIS 116847 (CTGCTAGCCTCTGGATTTGA, SEQ ID No: 48) and ISIS 116845(ACATAGCGCCTCTGACTGGG, SEQ ID No: 49). The mismatch control for ISIS116847 is ISIS 116848 (CTTCTGGCATCCGGTTTAGA, SEQ ID No: 50), a six basepair mismatch of ISIS 116847, while the universal control used is ISIS29848 (NNNNNNNNNNNNNNNNNNNN, SEQ ID No: 51) where N is a mixture of A,G, T and C. Both ISIS 116847 and ISIS 116845 target the coding region ofGenbank accession no. U93051, with ISIS 116847 starting at position 1063and ISIS 116845 starting at position 505.

These oligonucleotide sequences also target the mouse PTEN sequence withperfect complementarity, with ISIS 116845 targeting nucleotides1453-1472 and ISIS 116847 targeting nucleotides 2012-2031 of GenBankaccession no. U92437 (locus name MMU92437; Steck et al., Nature Genet.,1997, 15,356-362. Similarly, these oligonucleotide sequences target therat PTEN sequence with perfect complementarity, with ISIS 116845targeting nucleotides 505-524 and ISIS 116847 targeting nucleotides1063-1082 of GenBank accession no. AF017185.

All compounds are chimeric oligonucleotides (“gapmers”) nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotides. Allcytidine residues are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in otherexamples herein and are averaged from two experiments.

In a dose-response experiment, human hepatocyte cells (HEPG2; AmericanType Culture Collection, Manassas, Va.), mouse primary hepatocytes, andrat primary hepatocytes were treated with ISIS 116847 and its mismatchcontrol, ISIS 116848 at doses of 10, 50, 100 and 200 nM oligonucleotidenormalized to untreated controls. In all three species, the doseresponse was linear compared to vehicle treated controls.

In human HEPG2 cells, ISIS 116847 reduced PTEN mRNA levels to 55% ofcontrol at a dose of 10 nm, and to 5% of control at 200 nM while thePTEN mRNA levels in cells treated with the mismatch controloligonucleotide remained at greater than 90% of control across theentire dosing range.

In mouse primary hepatocytes the trend was the same with ISIS 116847reducing PTEN mRNA levels to 85% of control at the lower dose of 10 nM,and down to 2% of control at the 200 nM dose. Again, the controloligonucleotide, ISIS 116848 failed to reduce PTEN mRNA levels andremained at or above 85% of control.

In rat primary hepatocytes, ISIS 116847 reduced PTEN mRNA levels to 55%of control at the lower dose of 10 nM and to 10% of control at thehighest dose of 200 nM. PTEN mRNA levels in cells treated with thecontrol oligonucleotide, ISIS 116848, remained at or above 95% ofcontrol across the entire dosing range.

Example 19

Effects of Antisense Inhibition of PTEN on mRNA Expression in Fat andLiver

In the following examples, antisense inhibitors of PTEN are tested indb/db mice (Jackson Laboratories, Bar Harbor, Me.). These mice arehyperglycemic, obese, hyperlipidemic, and insulin resistant, and areused as a standard animal model of diabetes.

Male db/db mice (age 14 weeks) were divided into matched groups (n=5)with the same average blood glucose levels and treated once a week for 4weeks with ISIS 116847 at 10, 25, 50 or 100 mg/kg or ISIS 116845 at 50mg/kg. wild type mice were similarly treated. Controls included saline,ISIS 116848 (a mismatch control), ISIS 29848 (the universal controldiscussed in Example 18) and the sense control of ISIS 116847. As acomparison db/db mice were also treated with troglitazone, an oralantihyperglycemic agent which is used in the treatment of type IIdiabetes. It acts primarily to decrease insulin resistance, improvesensitivity to insulin in muscle and adipose tissue and inhibit hepaticgluconeogenesis. At day 28 mice were sacrificed and PTEN MRNA levelswere measured.

Treatment of db/db mice with ISIS 116847 showed a dose-dependentdecrease in PTEN mRNA levels in the liver to 10% of control at 50 mg/kg.ISIS 116845 showed a reduction in PTEN mRNA levels to 22% of control ata dose of 50 mg/kg.

In wild-type mice a level of 5% of control PTEN mRNA required a dose of100 mg/kg of ISIS 116847. Neither troglitazone nor any of the controlshad an effect on PTEN mRNA levels over saline control.

Similar results were seen in fat. Treatment of db/db mice with ISIS116847 showed a dose-dependent decrease in PTEN mRNA levels in fat to20% of control at 50 mg/kg. ISIS 116845 showed a reduction in PTEN mRNAlevels to 35% of control at a dose of 50 mg/kg.

In wild-type mice a level of 18% of control required a dose of 100 mg/kgof ISIS 116847. Neither troglitazone nor any of the controls had aneffect on PTEN mRNA levels over saline control.

In another experiment, male db/db mice (age 14 weeks) were divided intomatched groups (n=5) with the same average blood glucose levels andtreated intraperitoneally with saline or ISIS 116847 every other day(q2d) or twice a week (q4d) at a dose of 20 mg/kg. The control for bothprotocols was the mismatch control, ISIS 116848. Mice were exsanguinatedon day 14 and PTEN mRNA levels in liver and fat were measured.

ISIS 116847 successfully reduced PTEN mRNA levels in both liver and fatof db/db mice at both the q2d and q4d dosing schedules in adose-dependent manner, whereas the mismatch control and saline treatedanimals showed no reduction in PTEN mRNA.

There was no reduction of PTEN mRNA in skeletal muscle with any of theantisense oligonucleotides used. This lack of an effect in muscleindicates that reduction of expression of PTEN in liver and fat alone issufficient to lower hyperglycemia.

Example 20

Effects of Antisense Inhibition of PTEN on mRNA Expression in Kidney

Male db/db and wild-type mice were treated once a week for 4 weeks withISIS 116847 at 10, 25, 50 or 100 mg/kg or ISIS 116845 at 50 mg/kg.Controls included saline, ISIS 116848 (a mismatch control), ISIS 29848(the universal control discussed in Example 18) and the sense control ofISIS 116847. As a comparison db/db mice were also treated withtroglitazone. At day 28 mice were sacrificed and PTEN mRNA levels weremeasured.

Treatment with ISIS 116847 showed a dose-dependent decrease in PTEN mRNAlevels in kidney, being reduced to 70% of control at a dose of 50 mg/kg.ISIS 116845 reduced PTEN mRNA levels to 85% of control at the same dose.

In wild-type mice a level of 75% of control required a dose of loo mg/kgof ISIS 116847. Neither troglitazone nor any of the controls had aneffect on PTEN mRNA levels over saline control.

Example 21

Effects of Antisense Inhibition of PTEN (ISIS 116847) on PTEN ProteinLevels in Liver Extracts as a Function of Time and Dose

Male db/db and wild-type mice (age 14 weeks) were treated once a weekfor 4 weeks with saline, a control oligonucleotide, ISIS 29848 (50mg/kg) or ISIS 116847 at 10, or 50 mg/kg. Wild-type mice were treatedwith saline or ISIS 116847 at 100 mg/kg. Mice were sacrificed at day 28and PTEN protein levels were measured by Western blotting as describedin other examples herein.

In the db/db mice, treatment with ISIS 116847 caused a dose-dependentdecrease in PTEN protein levels compared to saline controls or mismatchtreated animals.

Protein levels in wild-type mice treated at 100 mg/kg were comparablyreduced to the levels seen in db/db mice treated at the 50 mg/kg dose.There was no significant difference in the relative levels of PTENprotein in control lean versus db/db mice.

Example 22

Effects of Antisense Inhibition of PTEN (ISIS 116847) on PTEN ProteinLevels in Fat and Kidney as a Function of Time and Dose

Male db/db and wild-type mice (age 14 weeks) were treated once a weekfor 4 weeks with saline or ISIS 116847 at 50 mg/kg by intraperitonealinjection. Mice were sacrificed at day 28 and PTEN protein levels weremeasured by Western blotting described in other examples herein.

PTEN levels in fat were reduced in both db/db and wildtype mice by thePTEN antisense as compared to control, and slight reduction of PTENlevels was seen in the kidney after antisense treatment.

Example 23

Effects of Antisense Inhibition of PTEN on Blood Glucose Levels

Male db/db and wild type mice (age 14 weeks) were divided into matchedgroups (n=5) with the same average blood glucose levels and treated byintraperitoneal injection with saline or ISIS 116847 every other day(q2d) or twice a week (q4d) at a dose of 20 mg/kg. The control for bothprotocols was the mismatch control, ISIS 116848. Blood glucose levelswere measured on day 7 and day 14.

By day 14 in db/db mice, blood glucose levels were reduced for bothtreatment schedules; from starting levels of 330 mg/dL to 175 mg/dL(q2d) and 170 mg/dL (q4d) which are levels within the range considerednormal for wild-type mice. The mismatch control levels remained at 310mg/dL throughout the study.

In wild-type mice, blood glucose levels remained constant throughout thestudy for all treatment groups (average 115 mg/dL).

In a similar experiment, male db/db and wild-type mice were treated oncea week for 4 weeks with ISIS 116847 or ISIS 116845 at 50 mg/kg. Controlsincluded saline, ISIS 116848 (a mismatch control) and ISIS 29848 (theuniversal control discussed in Example 18). At day 28 mice weresacrificed and serum glucose levels were measured.

In db/db mice, treatment with either ISIS 116847 or ISIS 116845 reducedserum glucose levels relative to saline control (480 mg/dL) to 240 and280 mg/dL, respectively. This reduction was statistically significant(p<0.005). Neither the mismatch nor universal control had any effect onserum glucose levels. In wild-type animals, ISIS 116847 failed to reduceserum glucose levels from that of control (200 mg/dL).

Example 24

Effects of Antisense Inhibition of PTEN (ISIS 116847) on Blood GlucoseLevels of db/db Mice as a Function of Time and Dose

Male db/db mice (age 14 weeks) were treated once a week for 4 weeks withsaline or ISIS 116847 at 10, 25 or 50 mg/kg intraperitoneally. Bloodglucose levels were measured on day 7, 14, 21 and 28.

At the beginning of the study, all groups had blood glucose levels of275 mg/dL which rose in the saline treated animals and those treated atthe low dose of ISIS 116847 to 350 mg/dL and 320 mg/dL, respectively byday four. At the end of the first week, all three dosing schedulesshowed a reduction in blood glucose and continued to show linear doseresponse decreases throughout the study. At day 28, blood glucose levelsin antisense treated animals were 275 mg/dL (10 mg/kg dose), 175 mg/dL(25 mg/kg dose) and 120 mg/dL (50 mg/kg dose) while saline treatedlevels remained at 350 mg/dL. The average glucose levels foroligonucleotide treated mice at the end of the four week study was 194mg/dL as compared to 418 mg/dL for saline treated controls (p<0.0001).

Example 25

Effects of Antisense Inhibition of PTEN (ISIS 116847) on Blood GlucoseLevels of db/db Mice-insulin Tolerance Test

Male db/db mice (age 14 weeks) were treated once with saline or ISIS116847 50 mg/kg by intraperitoneal injection. The insulin tolerance testwas performed after a four hour fast followed by an intraperitonealinjection of 1 U/kg human insulin (Lilly). On day 21, blood waswithdrawn from the tail at 0, 30, 60 and 90 minutes and blood glucoselevels were measured as a percentage of blood glucose at time zero.Statistical analysis was performed using ANOVA repeated measuresfollowed by Bonferroni Dunn analysis, p<0.05.

Treatment with ISIS 116847 on day 21 resulted in a significantdose-dependent decrease in blood glucose (p<0.006) at the 90 minutepost-treatment time point to 45% of control (55% decrease). Salinetreatment resulted in a 30% reduction. These studies indicate that thePTEN antisense oligonucleotide is capable of increasing sensitivity toinsulin (decreasing insulin resistance) and that treatment does notcause hypoglycemia. Glucose levels in PTEN treated mice (both db/db andwild-type) fasted for 16 hours remained normal.

Example 26

Effects of Antisense Inhibition of PTEN on Serum Triglyceride andCholesterol Concentration

Male db/db and wild-type mice were treated once a week for 4 weeks withISIS 116847 at 10, 25, 50 or 100 mg/kg or ISIS 116845 at 50 mg/kg.Controls included saline, ISIS 116848 (a mismatch control), ISIS 29848(the universal control discussed in Example 18) and the sense control ofISIS 116847. As a comparison db/db mice were also treated withtroglitazone. At day 28 mice were sacrificed and triglyceride andcholesterol levels were measured.

Treatment of db/db mice with ISIS 116847 resulted in a dose-dependentreduction of both triglycerides and cholesterol compared to salinecontrols (a reduction from 200 mg/dL to 100 mg/dL for triglycerides andfrom 130 mg/dL to 98 mg/dL for cholesterol). Treatment of db/db micewith ISIS 116845 at a dose of 50 mg/kg resulted in a decrease in bothtriglycerides and cholesterol levels to 130 mg/dL and 75 mg/dL,respectively. Troglitazone treatment of db/db mice reduced bothtriglyceride and cholesterol levels to 85 mg/dL each.

Wild-type animals did not respond to antisense treatment with ISIS116847 at a dose of 100 mg/kg as both triglyceride and cholesterollevels remained similar to control saline treated animals (between 85and 105 mg/dL). The reductions seen in cholesterol and triglycerideswere statistically significant at p<0.005.

Example 27

Effects of Antisense Inhibition of PTEN on Body Weight

Male db/db and wild-type mice were treated once a week for 4 weeks withISIS 116847 at 10, 25, 50 or 100 mg/kg or ISIS 116845 at 50 mg/kg.Controls included saline, ISIS 116848 (a mismatch control), ISIS 29848(the universal control discussed in Example 18) and the sense control ofISIS 116847. As a comparison db/db mice were also treated withtroglitazone. At day 28 mice were sacrificed and final body weights weremeasured.

Treatment with ISIS 116847 resulted in a dose-dependent increase in bodyweight over the dose range with animals treated at the high dose gainingan average of 8.7 grams while saline treated controls gained 2.8 grams.Animals treated with the mismatch or universal control oligonucleotidegained between 2.5 and 3.5 grams of body weight and troglitazone treatedanimals gained 5.0 grams.

Wild-type animals treated with 100 mg/kg of ISIS 116847 gained 2.0 gramsof body weight compared to a gain of 1.3 grams for the wild-type salineor mismatch controls.

Weight gain in the PTEN antisense treated mice began to increaserelative to that in saline or control groups at the same time thatglucose levels began to drop.

Example 28

Effects of Antisense Inhibition of PTEN on Liver Weight-anterior Lobe

Male db/db and wild-type mice were treated once a week for 4 weeks withISIS 116847 at 10, 25, 50 or 100 mg/kg or ISIS 116845 at 50 mg/kg.Controls included saline, ISIS 116848 (a mismatch control), ISIS 29848(the universal control discussed in Example 18) and the sense control ofISIS 116847. As a comparison db/db mice were also treated withtroglitazone. At day 28 mice were sacrificed and the weights of theanterior lobe of the liver were measured.

db/db animals treated at the high dose had liver weights of 1.2 gramswhile saline treated controls weighed 0.75 grams. db/db animals treatedwith ISIS 116845 at a dose of 50 mg/kg had comparable liver size tothose treated with ISIS 116847 at a dose of 25 mg/kg (1.0 grams).Animals treated with the mismatch control, universal control ortroglitazone had livers weighing an average of 1.0 gram.

Wild-type mouse livers treated with 100 mg/kg of ISIS 116847 weighed 0.7grams compared to 0.5 grams for the wildtype saline treated controls.

BrdU (bromine deoxyuridine) staining of of liver sections indicated thatthe increase in liver weight was not due to increased cellproliferation, and there was no increase in inflammatory infiltrates inthe liver. Long-term studies show that the increases in liver weight arereversed.

Example 29

Effects of Antisense Inhibition of PTEN (ISIS 116847) on PEPCK mRNAExpression in Liver of db/db Mice

PEPCK is the rate-limiting enzyme of gluconeogenesis and is expressedpredominantly in liver where it acts in the gluconeogenic pathway(production of glucose from amino acids) and in kidney where it acts inthe gluconeogenic pathway as well as being glyceroneogenic andammoniagenic. In the liver, PEPCK is negatively regulated by insulin andhas therefore been considered a potential contributing factor tohyperglycemia in diabetics (Sutherland et al., Philos. Trans. R. Soc.Lond. B. Biol. Sci., 1996, 351, 191-199).

Male db/db mice (age 14 weeks) with the same average blood glucoselevels were divided into groups (n=5) and treated intraperitoneally withsaline, ISIS 116847 or the mismatch control, ISIS 116848, every otherday (q2d). Mice were exsanguinated on day 14 and PEPCK mRNA levels inliver were measured.

Mice treated with ISIS 116847 showed a reduction of PEPCK mRNA to 65% ofsaline treated controls. The mismatch control group remained at 98% ofsaline treated control.

Example 30

Effects of Antisense Inhibition of PTEN (ISIS 116847) on Serum InsulinLevels of db/db Mice

Male db/db and wild type mice (age 14 weeks) were divided into matchedgroups (n=5) with the same average blood glucose levels and treated byintraperitoneal injection with saline or ISIS 116847 every other day(q2d) or twice a week (q4d) at a dose of 20 mg/kg. The control for bothprotocols was the mismatch control, ISIS 116848. Mice were exsanguinatedon day 14 and serum insulin levels were measured.

On day 14 db/db mice treated on the q2d schedule had serum insulinlevels of 7.8 ng/mL, compared to saline treated (9 ng/mL) and mismatchtreated animals (12 ng/mL). In the q4d schedule there was a drop in theserum insulin levels of db/db mice treated with ISIS 116847 to 4 ng/mLwhile the mismatch control levels remained at 12 ng/mL. Wild-type micehad serum insulin levels of 1 ng/mL throughout the course of bothtreatment schedules.

Example 31

Effects of Antisense Inhibition of PTEN on Liver Function-AST/ALT Levels

Male db/db and wild type mice (age 14 weeks) were divided into matchedgroups (n=5) with the same average blood glucose levels and treated byintraperitoneal injection with saline, troglitazone, or ISIS 116847every other day (q2d) or twice a week (q4d) at a dose of 20 mg/kg. Thecontrol for both protocols was the mismatch control, ISIS 116848. Micewere exsanguinated on day 14 and liver enzyme levels were measured.

In the q2d treatment schedule there was an increase in ALT levels oversaline treated animals from 125 IU/L (saline control) to 300 IU/L (bothPTEN oligonucleotide, ISIS 116847, and mismatch control), whereas ASTlevels remained between 220 IU/L and 240 IU/L among the three treatmentgroups.

In the q4d treatment schedule, ALT levels increased from 125 IU/L(saline control) to 160 IU/L in animals treated with ISIS 116847 and 200IU/L for mismatch control. AST levels decreased from saline controllevels of 220 IU/L to 160 IU/L for ISIS 116847 treated animals, as wellas in animals treated with the mismatch control (200 IU/L). As acomparison, AST and ALT levels were measured after treatment withtroglitazone. Levels of both enzymes were found to be 260 IU/L.

In a similar experiment, male db/db and wild-type mice were treated oncea week for 4 weeks with ISIS 116847 at 10, 25, 50 or 100 mg/kg or ISIS116845 at 50 mg/kg. Controls included saline or ISIS 29848 (theuniversal control discussed in Example 18). As a comparison db/db micewere also treated with troglitazone. At day 28 mice were sacrificed andAST and ALT levels were measured.

Treatment of db/db mice with ISIS 116847 resulted in a dose-dependentincrease in ALT levels over the dose range with animals treated at thehigh dose having ALT levels of 250 IU/L while AST levels remainedconstant at 165 IU/L. These levels represent an increase in ALT levelsfrom saline treated controls of 110 IU/L and a decrease in AST levelsfrom saline treated controls of 220 IU/L. db/db animals treated withISIS 116845 at a dose of 50 mg/kg had comparable ALT and AST levels, 145IU/L. Animals treated with the universal control had ALT and AST levelscomparable to control levels and those treated with troglitazone showedan increase in ALT levels over control to 150 IU/L and a slight decreasein AST levels to 200 IU/L from control.

Wild-type mice treated with 100 mg/kg of ISIS 116847 had both increasedALT and AST levels (100 IU/L and 130 IU/L, respectively) compared tosaline-treated control ALT and AST levels (50 IU/L and 95 IU/L,respectively).

Although ALT levels were slightly elevated in PTEN antisense treatedanimals, AST levels were reduced indicating that PTEN antisense effectson liver weight were not due to toxicity.

51 1 1212 DNA Homo sapiens CDS (1)..(1212) 1 atg aca gcc atc atc aaa gagatc gtt agc aga aac aaa agg aga tat 48 Met Thr Ala Ile Ile Lys Glu IleVal Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 caa gag gat gga ttc gac ttagac ttg acc tat att tat cca aac att 96 Gln Glu Asp Gly Phe Asp Leu AspLeu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 att gct atg gga ttt cct gca gaaaga ctt gaa ggc gta tac agg aac 144 Ile Ala Met Gly Phe Pro Ala Glu ArgLeu Glu Gly Val Tyr Arg Asn 35 40 45 aat att gat gat gta gta agg ttt ttggat tca aag cat aaa aac cat 192 Asn Ile Asp Asp Val Val Arg Phe Leu AspSer Lys His Lys Asn His 50 55 60 tac aag ata tac aat ctt tgt gct gaa agacat tat gac acc gcc aaa 240 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg HisTyr Asp Thr Ala Lys 65 70 75 80 ttt aat tgc aga gtt gca caa tat cct tttgaa gac cat aac cca cca 288 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe GluAsp His Asn Pro Pro 85 90 95 cag cta gaa ctt atc aaa ccc ttt tgt gaa gatctt gac caa tgg cta 336 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp LeuAsp Gln Trp Leu 100 105 110 agt gaa gat gac aat cat gtt gca gca att cactgt aaa gct gga aag 384 Ser Glu Asp Asp Asn His Val Ala Ala Ile His CysLys Ala Gly Lys 115 120 125 gga cga act ggt gta atg ata tgt gca tat ttatta cat cgg ggc aaa 432 Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu LeuHis Arg Gly Lys 130 135 140 ttt tta aag gca caa gag gcc cta gat ttc tatggg gaa gta agg acc 480 Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr GlyGlu Val Arg Thr 145 150 155 160 aga gac aaa aag gga gta act att ccc agtcag agg cgc tat gtg tat 528 Arg Asp Lys Lys Gly Val Thr Ile Pro Ser GlnArg Arg Tyr Val Tyr 165 170 175 tat tat agc tac ctg tta aag aat cat ctggat tat aga cca gtg gca 576 Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu AspTyr Arg Pro Val Ala 180 185 190 ctg ttg ttt cac aag atg atg ttt gaa actatt cca atg ttc agt ggc 624 Leu Leu Phe His Lys Met Met Phe Glu Thr IlePro Met Phe Ser Gly 195 200 205 gga act tgc aat cct cag ttt gtg gtc tgccag cta aag gtg aag ata 672 Gly Thr Cys Asn Pro Gln Phe Val Val Cys GlnLeu Lys Val Lys Ile 210 215 220 tat tcc tcc aat tca gga ccc aca cga cgggaa gac aag ttc atg tac 720 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg GluAsp Lys Phe Met Tyr 225 230 235 240 ttt gag ttc cct cag ccg tta cct gtgtgt ggt gat atc aaa gta gag 768 Phe Glu Phe Pro Gln Pro Leu Pro Val CysGly Asp Ile Lys Val Glu 245 250 255 ttc ttc cac aaa cag aac aag atg ctaaaa aag gac aaa atg ttt cac 816 Phe Phe His Lys Gln Asn Lys Met Leu LysLys Asp Lys Met Phe His 260 265 270 ttt tgg gta aat aca ttc ttc ata ccagga cca gag gaa acc tca gaa 864 Phe Trp Val Asn Thr Phe Phe Ile Pro GlyPro Glu Glu Thr Ser Glu 275 280 285 aaa gta gaa aat gga agt cta tgt gatcaa gaa atc gat agc att tgc 912 Lys Val Glu Asn Gly Ser Leu Cys Asp GlnGlu Ile Asp Ser Ile Cys 290 295 300 agt ata gag cgt gca gat aat gac aaggaa tat cta gta ctt act tta 960 Ser Ile Glu Arg Ala Asp Asn Asp Lys GluTyr Leu Val Leu Thr Leu 305 310 315 320 aca aaa aat gat ctt gac aaa gcaaat aaa gac aaa gcc aac cga tac 1008 Thr Lys Asn Asp Leu Asp Lys Ala AsnLys Asp Lys Ala Asn Arg Tyr 325 330 335 ttt tct cca aat ttt aag gtg aagctg tac ttc aca aaa aca gta gag 1056 Phe Ser Pro Asn Phe Lys Val Lys LeuTyr Phe Thr Lys Thr Val Glu 340 345 350 gag ccg tca aat cca gag gct agcagt tca act tct gta aca cca gat 1104 Glu Pro Ser Asn Pro Glu Ala Ser SerSer Thr Ser Val Thr Pro Asp 355 360 365 gtt agt gac aat gaa cct gat cattat aga tat tct gac acc act gac 1152 Val Ser Asp Asn Glu Pro Asp His TyrArg Tyr Ser Asp Thr Thr Asp 370 375 380 tct gat cca gag aat gaa cct tttgat gaa gat cag cat aca caa att 1200 Ser Asp Pro Glu Asn Glu Pro Phe AspGlu Asp Gln His Thr Gln Ile 385 390 395 400 aca aaa gtc tga 1212 Thr LysVal 2 26 DNA Artificial Sequence PCR Primer 2 aatggctaag tgaagatgacaatcat 26 3 25 DNA Artificial Sequence PCR Primer 3 tgcacatatcattacaccag ttcgt 25 4 30 DNA Artificial Sequence PCR Probe 4 ttgcagcaattcactgtaaa gctggaaagg 30 5 19 DNA Artificial Sequence PCR Primer 5gaaggtgaag gtcggagtc 19 6 20 DNA Artificial Sequence PCR Primer 6gaagatggtg atgggatttc 20 7 20 DNA Artificial Sequence PCR Probe 7caagcttccc gttctcagcc 20 8 18 DNA Artificial Sequence AntisenseOligonucleotide 8 cgagaggcgg acgggacc 18 9 18 DNA Artificial SequenceAntisense Oligonucleotide 9 cgggcgcctc ggaagacc 18 10 18 DNA ArtificialSequence Antisense Oligonucleotide 10 tggctgcagc ttccgaga 18 11 18 DNAArtificial Sequence Antisense Oligonucleotide 11 cccgcggctg ctcacagg 1812 18 DNA Artificial Sequence Antisense Oligonucleotide 12 caggagaagccgaggaag 18 13 18 DNA Artificial Sequence Antisense Oligonucleotide 13gggaggtgcc gccgccgc 18 14 18 DNA Artificial Sequence AntisenseOligonucleotide 14 atggtgacag gcgactca 18 15 18 DNA Artificial SequenceAntisense Oligonucleotide 15 ccgggtccct ggatgtgc 18 16 18 DNA ArtificialSequence Antisense Oligonucleotide 16 cctccgaacg gctgcctc 18 17 18 DNAArtificial Sequence Antisense Oligonucleotide 17 tctcctcagc agccagag 1818 18 DNA Artificial Sequence Antisense Oligonucleotide 18 cgcttggctctggaccgc 18 19 18 DNA Artificial Sequence Antisense Oligonucleotide 19tcttctgcag gatggaaa 18 20 18 DNA Artificial Sequence AntisenseOligonucleotide 20 tgctaacgat ctctttga 18 21 18 DNA Artificial SequenceAntisense Oligonucleotide 21 ggataaatat aggtcaag 18 22 18 DNA ArtificialSequence Antisense Oligonucleotide 22 tcaatattgt tcctgtat 18 23 18 DNAArtificial Sequence Antisense Oligonucleotide 23 ttaaatttgg cggtgtca 1824 18 DNA Artificial Sequence Antisense Oligonucleotide 24 caagatcttcacaaaagg 18 25 18 DNA Artificial Sequence Antisense Oligonucleotide 25attacaccag ttcgtccc 18 26 18 DNA Artificial Sequence AntisenseOligonucleotide 26 tgtctctggt ccttactt 18 27 18 DNA Artificial SequenceAntisense Oligonucleotide 27 acatagcgcc tctgactg 18 28 18 DNA ArtificialSequence Antisense Oligonucleotide 28 tgtgaaacaa cagtgcca 18 29 18 DNAArtificial Sequence Antisense Oligonucleotide 29 gaatatatct tcaccttt 1830 18 DNA Artificial Sequence Antisense Oligonucleotide 30 ggaagaactctactttga 18 31 18 DNA Artificial Sequence Antisense Oligonucleotide 31tgaagaatgt atttaccc 18 32 18 DNA Artificial Sequence AntisenseOligonucleotide 32 atttcttgat cacataga 18 33 18 DNA Artificial SequenceAntisense Oligonucleotide 33 ggttggcttt gtctttat 18 34 18 DNA ArtificialSequence Antisense Oligonucleotide 34 tgctagcctc tggatttg 18 35 18 DNAArtificial Sequence Antisense Oligonucleotide 35 tctggatcag agtcagtg 1836 18 DNA Artificial Sequence Antisense Oligonucleotide 36 tattttcatggtgtttta 18 37 18 DNA Artificial Sequence Antisense Oligonucleotide 37tgttcctata actggtaa 18 38 18 DNA Artificial Sequence AntisenseOligonucleotide 38 gtgtcaaaac cctgtgga 18 39 18 DNA Artificial SequenceAntisense Oligonucleotide 39 actggaataa aacgggaa 18 40 18 DNA ArtificialSequence Antisense Oligonucleotide 40 acttcagttg gtgacaga 18 41 18 DNAArtificial Sequence Antisense Oligonucleotide 41 tagcaaaacc tttcggaa 1842 18 DNA Artificial Sequence Antisense Oligonucleotide 42 aattatttcctttctgag 18 43 18 DNA Artificial Sequence Antisense Oligonucleotide 43taaatagctg gagatggt 18 44 18 DNA Artificial Sequence AntisenseOligonucleotide 44 cagattaata actgtagc 18 45 18 DNA Artificial SequenceAntisense Oligonucleotide 45 ccccaataca gattcact 18 46 18 DNA ArtificialSequence Antisense Oligonucleotide 46 attgttgctg tgtttctt 18 47 18 DNAArtificial Sequence Antisense Oligonucleotide 47 tgtttcaagc ccattctt 1848 20 DNA Artificial Sequence Antisense Oligonucleotide 48 ctgctagcctctggatttga 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49acatagcgcc tctgactggg 20 50 20 DNA Artificial Sequence AntisenseOligonucleotide 50 cttctggcat ccggtttaga 20 51 20 DNA ArtificialSequence unsure (1)..(20) Antisense Oligonucleotide 51 nnnnnnnnnnnnnnnnnnnn 20

What is claimed is:
 1. A compound target to a nucleic acid moleculeencoding PTEN wherein the compound is an antisense oligonucleotidehaving a sequence consisting of SEQ ID NOS: 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 48, or
 49. 2. The compound of claim1, wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 3. The compound of claim 2, wherein themodified internucleoside linkage is a phosphorothioate linkage.
 4. Thecompound of claim 1, wherein the antisense oligonucleotide comprises atleast one modified sugar moiety.
 5. The compound of claim 4, wherein themodified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 6. Thecompound of claim 1, wherein the antisense oligonucleotide comprises atleast one modified nucleobase.
 7. The compound of claim 6, wherein themodified nucleobase is a 5-methylcytosine.
 8. The compound of claim 1,wherein the antisense oligonucleotide is a chimeric oligonucleotide. 9.A composition comprising the compound of claim 1, and a pharmaceuticallyacceptable carrier or diluent.
 10. The composition of claim 9, furthercomprising a colloidal dispersion system.
 11. A method of inhibiting theexpression of PTEN in cells or tissues comprising contacting said cellsor tissues in vitro with the compound of claim 1, so that expression ofPTEN is inhibited.
 12. The method of claim 11, wherein the cells ortissues are human cells or tissues.
 13. The method of claim 11, whereinthe cells or tissues are rodent cells or tissues.
 14. The method ofclaim 13, wherein the rodent cells or tissues are mouse cells ortissues.
 15. The method of claim 13, wherein the rodent cells or tissuesare rat cells or tissues.
 16. The method of claim 11, wherein the cellsor tissues are liver, kidney or adipose cells or tissues.
 17. A methodof treating an animal having diabetes associated with PTEN expressionscomprising administering to said diabetic animal a therapeutically orprophylactically effective amount of an antisense oligonucleotide havinga sequence consisting of SEQ ID NO:48 or SEQ ID NO:49 so that expressionof PTEN is inhibited and blood glucose levels of said animal aredecreased.
 18. The method of claim 17 wherein the animal is a human orrodent.
 19. The method of claim 17, wherein the diabetes is Type 2diabetes.
 20. The method of claim 17, wherein the blood glucose levelsare plasma glucose levels or serum glucose levels.
 21. A method ofinhibiting expression of PEPCK, phosphoenolpyruvate carboxykinase, incells or tissues from a diabetic animal comprising contacting said cellsor tissues from a diabetic animal with an antisense oligonucleotidehaving a sequence consisting of SEQ ID NO:48 or SEQ ID NO:49 so thatboth expression of PEPCK and PTEN are inhibited.
 22. A method ofdecreasing blood insulin levels in a diabetic animal comprisingadministering to said diabetic animal an antisense oligonucleotidehaving a sequence consisting of SEQ ID NO:48 or SEQ ID NO:49 whereinexpression of PTEN is inhibited and blood insulin levels are decreased.23. The method of claim 22, wherein the animal is a human or a rodent.24. The method of claim 22, wherein the blood insulin levels plasmainsulin levels or serum insulin levels.
 25. A method of decreasinginsulin resistance in a diabetic animal comprising administering to saiddiabetic animal an antisense oligonucleotide having a sequenceconsisting of SEQ ID NO:48 or SEQ ID NO:49 wherein expression of PTEN isinhibited and insulin resistance is decreased.
 26. The method of claim25, wherein the animal is a human or a rodent.
 27. A method ofincreasing insulin sensitivity in a diabetic animal comprisingadministering to said diabetic animal an antisense oligonucleotidehaving a sequence consisting of SEQ ID NO:48 or SEQ ID NO:49 whereinexpression of PTEN is inhibited and insulin sensitivity is increased.28. The method of claim 27, wherein the animal is a human or a rodent.29. A method of decreasing blood triglyceride levels in a diabeticanimal comprising administering to said diabetic animal an antisenseoligonucleotide having a sequence consisting of SEQ ID NO:48 or SEQ IDNO:49 wherein expression of PTEN is inhibited and blood triglyceridelevels are decreased.
 30. The method of claim 29, wherein the animal isa human or a rodent.
 31. A method of decreasing blood cholesterol levelsin a diabetic animal comprising administering to said diabetic animal anantisense oligonucleotide having a sequence consisting of SEQ ID NO:48or SEQ ID NO:49 wherein expression of PTEN is inhibited and bloodcholesterol levels are decreased.
 32. The method of claim 31, whereinthe animal is a human or a rodent.